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F. Guillem et al.
resented orthochromatic erythroblasts and reticulocytes, as previously described,3 we sorted them by FACS based on their Band3 and 4-integrin expression (Figure 4C). As expected, mature erythroblast content (orthochromatic erythroblasts+reticulocytes) was enriched in high Band3 gate (62.23%±5.31) compared to low Band3 gate (22.83%±3.49) (P=0.0004).
GAP1) essential for XPO1 activity are also expressed dur- ing erythroid differentiation and also down-regulated at the time of caspase activation (data not shown). Furthermore, we demonstrate that inhibition of XPO1 by KPT-251 treatment improves terminal maturation of β- TM erythroblasts by preventing GATA-1 degradation by caspase-3. Thus, XPO1 inhibitors could be added to the armamentarium of therapeutic options of β-TM to cor- rect ineffective erythropoiesis and its pathological conse- quences (e.g. iron overload and extramedullary hematopoiesis), to increase hemoglobin level, to reduce transfusion burden and ultimately to increase overall sur- vival. Interestingly, KPT compounds are currently under clinical development and exhibit high efficacy and safety profile in cancer therapy. More recently, a new generation of KPT compounds with reduced toxicity is being devel- oped (clinicaltrials.gov identifier: NCT02649790) and as such, if shown to be not toxic in the long-term, they could be used in β-TM as well. In addition, maintaining HSP70 in the nucleus by blocking XPO1 could be useful in other pathologies of erythropoiesis where HSP70 is not localized in the nucleus, such as myelodysplastic syn- drome25 and congenital erythroblastopenia,26 but also in other diseases such as some neurodegenerative diseases, in which protein aggregates may also prevent HSP70 nuclear location.
Acknowledgments
The authors would like to thank the Department of Biotherapy and the Maternity at the Necker Hospital (Paris, France) for pro- viding blood samples. This work was supported by the French National Research Agency ANR-13-BSV1-0029-05 “HSPathies”, LabEx GRex financial support ANR-11-IDEX-0, the French National Research Agency (ANR) under the program “Investissements d’Avenir” (LabExGRex and LipSTIC), the Institut National du Cancer (INCa), the Ligue Nationale Contre le Cancer (“Label of Excellence”), Ministère de l'Enseignement Supérieur et de la Recherche, FEDER and Région Bourgogne.
Funding
This program has received a state subsidy managed by the National Research Agency under the "Investments for the Future" program bearing the reference ANR-01-A0-IAHU.
We further confirmed that in β-TM progenitors, KPT treatment alleviated the maturation arrest at the polychro- matophilic stage as it decreased the proportion of poly- chromatophilic cells and increased the proportion of mature cells (Figure 4D). More importantly, KPT treat- ment significantly increased the terminal maturation index (TMI), determined as the ratio of orthochromatic erythroblasts+reticulocytes *100/ polychromatophilic cells, at the dose of 1000nM KPT [TMI normalized to con- trol DMSO: DMSO=1, KPT100nM=1.8±0.5 (NS) KPT1000nM=3.7±1.05, (P<0.05)] (Figure 4E). Finally, we show that the significant increase in TMI and in absolute number of mature cells at 1000nM is associated with a high increase in HbF amount in the pool of mature cells, which is not observed at lower doses of treatment. This effect was observed with a greater magnitude at higher dose (1500nM). However at this dose, inhibition of cell proliferation and apoptosis were higher (Online Supplementary Figure S4).
In Figure 5, we schematically illustrate the molecular mechanisms modulated by KPT treatment in β-TM ery- throid progenitors compared to β-TM and normal ery- throid progenitors in normal conditions.
Discussion
Taken together, our data demonstrate that XPO1 is the main nuclear exporter of HSP70 in various cell types, and it may participate in the regulation of HSP70 in human erythroblasts during normal and pathological erythro- poiesis. Decreased expression of XPO1 along erythroid differentiation provides an explanation for the augmenta- tion of nuclear localization of HSP70 during normal ery- throid maturation. Interestingly, proteins of the Ran machinery (RAN, RANBP1, RCC1, NUTF1 AND RAN-
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